Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 23)

7.8 Refractionation
Better separation may be achieved by refractionating the sample. The mini Rotofor
is ideal for refractionation because samples are minimally diluted in its chamber. After
analyzing the fractions from the initial separation, the fractions containing the protein of
interest should be diluted in distilled water and reloaded in the standard Rotofor cell or
the Mini Rotofor cell. Upon refractionation the ampholyte concentration should be at
least 0.5%. We recommend that no less than 4–5 fractions be pooled and reapplied for
a second Rotofor run. If urea or non-ionic detergents are needed to maintain protein
solubility add the same concentration as used in the first fractionation. Do not add
additional ampholytes or salts at this stage.
1. Dilute pooled fractions appropriately, e.g., with water, up to 8 M urea, or a solution
containing non-ionic detergent for solubility, to a final volume of 55–60 ml in the
standard Rotofor or 18 ml for the Mini Rotofor. The customized ampholyte blend
obtained will span the pI of the protein of interest. Do not add additional ampholyte
to the refractionation mix; the amount present in the pooled samples is suitable for
focusing and provides a narrow range pH gradient to increase separation of the
protein of interest.
2. Load the diluted sample and re-run. Since the ionic strength of the sample will
be lower upon refractionation, higher voltages, yielding better separations can
be achieved. Refractionations of low ionic strength solutions have been carried
out at 2,000–3000 volts. Do not exceed the power limit of the cell. Focusing is
usually complete in 3–5 hours. The upper limit for voltage is dependent on how
well heat can be dissipated. Set the coolant temperature between -5°C and -10°C
for high voltage separations.
Section 8
Analysis of Results
8.1 Fraction Analysis
After harvesting, it is important to analyze the fractions to determine which contain
the protein of interest. There are many different ways of doing this, and the best
method is dependent on the protein being analyzed.
SDS-PAGE analysis or an IEF gel, usually pH 3-10, will give an accurate
representation of the fractionation. Other methods for assaying which channels
contain the protein of interest are dependent on the particular protein and include
activity assays and antibody tests. Analytical gels should be silver stained for high
sensitivity detection of contaminants.
8.2 Separation of Ampholytes From Proteins
Many applications can tolerate the presence of ampholytes in protein solutions.
However, ampholytes can interfere with some assays such as amino acid analysis.
Several methods for separating ampholytes from focused proteins are listed below.
1. Preparative Electrophoresis - Rotofor fractions containing the protein of interest
and any remaining contaminating proteins can be pooled and applied to a
preparative continuous-elution electrophoresis cell such as Bio-Rad’s Model
491 Prep Cell. Using the Model 491 Prep Cell as second and a final purification
step, samples (Rotofor fractions) are electrophoresed through a polyacrylamide
gel. In this way, the contaminating proteins and the ampholytes are effectively
separated from the protein of interest.
19